This invention relates to the design and synthesis of nuclease resistant phosphorothioate oligonucleotides which are useful for therapeutics, diagnostics and as research reagents. Phosphorothioate oligonucleotides are provided in which all of the internucleoside linkages are chiral. Such compounds are resistant to nuclease degradation and are capable of modulating the activity of DNA and RNA.
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. For disease states, classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect can be obtained with minimal side effects. It is therefore a general object of such therapeutic approaches to interfere with or other-wise modulate gene expression, which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence. Several oligonucleotides are currently undergoing clinical trials for such use. Phosphorothioate oligonucleotides are presently being used as therapeutic agents in human clinical trials against various disease states, including use as antiviral agents.
In addition to such use as both indirect and direct regulators of proteins, oligonucleotides also have found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligomeric compounds via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides, both natural and synthetic, are employed as primers in such PCR technology.
Oligonucleotides are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et al., Eds., Current Publications, 1993. Such uses include as synthetic oligonucleotide probes, in screening expression libraries with antibodies and oligomeric compounds, DNA sequencing, in vitro amplification of DNA by the polymerase chain reaction, and in site-directed mutagenesis of cloned DNA. See Book 2 of Molecular Cloning, A Laboratory Manual, supra. See also xe2x80x9cDNA-protein interactions and The Polymerase Chain Reactionxe2x80x9d in Vol. 2 of Current Protocols In Molecular Biology, supra.
A number of chemical modifications have been introduced into oligonucleotides to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include those designed to increase binding to a target strand (i.e. increase melting temperatures, Tm), to assist in identification of an oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.
The complementarity of oligonucleotides has been used for inhibition of a number of cellular targets. Complementary oligonucleotides are commonly described as being antisense oligonucleotides. Various reviews describing the results of these studies have been published including Progress In Antisense Oligonucleotide Therapeutics, Crooke, S. T. and Bennett, C. F., Annu. Rev. Pharmacol. Toxicol., 1996, 36, 107-129. These oligonucleotides have proven to be powerful research tools and diagnostic agents. Certain oligonucleotides that have been shown to be efficacious are currently in human clinical trials.
The pharmacological activity of oligonucleotides, like other therapeutics, depends on a number of factors that influence the effective concentration of these agents at specific intracellular targets. One important factor for oligonucleotides is the stability of the species in the presence of nucleases. It is unlikely that unmodified, naturally-occurring oligonucleotides will be useful therapeutic agents because they are rapidly degraded by nucleases. The limitations of available methods for modification of the phosphate backbone of unmodified oligonucleotides have led to a continuing and long felt need for other modifications which provide resistance to nucleases and satisfactory hybridization properties for antisense oligonucleotide diagnostics and therapeutics.
Oligonucleotides having phosphorothioate modified backbones have shown therapeutic effects against numerous targets. This success is due in part to the increased nuclease resistance of the phosphorothioate backbone relative to the naturally occurring phosphodiester backbone. The phosphorothioate linkage unlike the phosphodiester linkage has 2 enantiomers, Rp and Sp. It has been shown that a 3xe2x80x2-Rp linkage is labile to at least one exonuclease in the cytosol of HUVEC cells (Kiziolkiewicz et al. Nucleosides and Nucleotides, 1997, vol. 16, pp. 1677-1682). See also Koziolkiewicz et al., Antisense Nucleic Acid Drug Dev., 1997, 7, 43-48; Koziolkiewicz, Maria, Gendaszewska, Edyta, Maszewska, Maria, Stability of Stereoregular Oligo(nucleoside phosphorothioate)s in Human Cells; Diastereoselectivity of Cellular 3xe2x80x2-Exonuclease, Nucleosides Nucleotides 1997, 16(7-9) 1677-1682.
A specific feature of oligonucleotides as drugs is that they must be stable in vivo long enough to be effective. Consequently, much research has been focused on enhancing the stability of oligonucleotide therapeutics while maintaining their specific binding properties. Recently, several groups have reported that chiral phosphorothioate oligonucleotide analogs have enhanced binding properties (Rp isomer) to the target RNA as well as significant stabilization to exonucleases (Sp isomer) (See Koziolkiewicz et al., Antisense and Nucleic acid drug development, 1997, 7, 43-8; Burgers et al., J. Biol. Chem., 1979, 254, 6889-93; and Griffiths et al., Nucleic Acids Research, 1987, 15, 4145-62).
Presently, there is no method to prepare P-chiral oligonucleotides in large scale. Current methods include synthesis and chromatographic isolation of stereoisomers of the chiral building blocks. (Stec et al., Angew. Chem. Int. Ed. Engl., 1994, 33, 709; Stec et al., J. Am. Chem. Soc., 1995, 117, 12019; and Stec W. J., Protocols for Oligonucleotides and Analogs: Synthesis and Properties, edited by Sudhir Agrawal, p. 63-80, (1993, Humana Press) and references cited therein). This method suffers from the non-stereospecific synthesis of the synthon. Recently, Just and coworkers presented the use of a chiral auxiliary to form dinucleotide phosphorothioate triesters in 97% ee (Wang, J. C., and Just G., Tetrahedron Letters, 1997, 38, 705-708). However, there was reported difficulty in removing the chiral auxiliary protecting group at phosphorous. This method has yet to be tested for convenient large scale automated synthesis.
Stereoregular phosphorothioate analogs of pentadecamer 5xe2x80x2-d(AGATGTTTGA GCTCT)-3xe2x80x2 were synthesized by the oxathiaphospholane method (Koziolkiewicz et al., Nucleic Acids Res., 1995, 23, 5000-5005). There diastereomeric purity was assigned by means of enzymic degradation with nuclease P1 and independently, with snake venom phosphodiesterase. DNA-RNA hybrids formed by phosphorothioate oligonucleotides (PS-oligos) with the corresponding complementary pentadecarbonucleotide were treated with bacterial RNase H. The DNA-RNA complex containing the PS-oligo of [all-RP] configuration was found to be more susceptible to RNase H-dependent degradation of the pentadecarbonucleotide compared with hybrids containing either the [all-SP] counterpart or the so called ""random mixture of diastereomers of the pentadeca(nucleoside phosphorothioate). This stereodependence of RNase H action was also observed for a polyribonucleotide (475 nt) hybridized with these phosphorothioate oligonucleotides. The results of melting studies of PS-oligo-RNA hybrids allowed a rationalization of the observed stereodifferentiation in terms of the higher stability of heterodimers formed between oligoribonucleotides and [all-RP]-oligo(nucleoside phosphorothioates), compared with the less stable heterodimers formed with [all-SP]-oligo(nucleoside phosphorothioates) or the random mixture of diastereomers.
(S)-1-(indol-2-yl)-propan-2-ol was used as a chiral auxiliary to form a dinucleotide phosphorothioate triester in 97% ee (Wang et al., Tetrahedron Lett., 1997, 38, 705-708).
A stereoselective preparation of dinucleotide hosphorothioates with a diastereomeric excess of  greater than 98%, using hydroxy(indolyl)butyronitrile I as chiral auxiliaries, is reported (Wang et al., Tetrahedron Lett., 1997, 38, 3797-3800).
1,2-O-Cyclopentylidene-5-deoxy-5-isopropylamino-D-xylofuranose and its enantiomer were used as chiral auxiliaries to form, respectively, Sp and Rp dithymidine phosphorothioates in 98% diastereomeric excess, using phosphoramidite methodologies and 2-bromo-4,5-dicyanoimidazole as catalyst (Jin et al., J. Org. Chem., 1998, 63, 3647-3654).
Oligonucleotide phosphorothioates were synthesized using prokaryotic DNA polymerase and oligonucleotide template/primer (Lackey et al., Biotechnol. Lett., 1997, 19, 475-478). The method facilitates the recovery of DNA polymerase and template/primer and is successful at the milligram scale. Thus, reusable template/primers were designed to specify the synthesis of an oligonucleotide (GPs0193) complementary to a sequence in exon 7 of the human immunodeficiency virus genome. Extension of the 3xe2x80x2-terminus by DNA polymerase utilizing dNTPS(Rp+Sp) substrates produced the specified oligonucleotide phosphorothioate with the chirally pure (Rp) stereochem. The biochemical synthesis was essentially complete within 60 min (compared with 24 h for automated solid phase synthesis), and produced  less than 5% intermediate length oligonucleotide products, corresponding to a stepwise yield of  greater than 99.7% for the addition of each nucleotide.
Phosphorothioate oligodeoxyribonucleotides were tested for their ability to recognize double-helical DNA in two distinct triple helix motifs (Hacia et al., Biochemistry, 1994, 33, 5367-5369). Purine-rich oligonucleotides containing a diastereomeric mixture of phosphorothioate or stereoregular (all Rp) phosphorothioate linkages are shown to form triple-helical complexes with affinities similar to those of the corresponding natural phosphodiester oligonucleotides. In contrast, pyrimidine-rich phosphorothioate oligonucleotides containing a mixture of diastereomeric or stereoregular (all Rp) linkages do not bind to double-helical DNA with measurable affinity. These observations have implications for triple helix structure and for biological applications.
An enzymatic protocol has been established for the synthesis of Stereoregular (all Rp) oligodeoxyribonucleotide phosphorothioates. A 25-mer oligodeoxynucleotide phosphorothioate has been synthesized and studied for biophysical and biochemical properties (Tang et al., Nucleosides and Nucleotides, 1995, 14, 985-990).
Stability of oligo(nucleoside phosphorothioate)s (PS-oligos) in HUVEC (human umbilical vein endothelial cells) has been studied (Koziolkiewicz et al., Nucleosides and Nucleotides, 1997, 16, 1677-1682). Cytosolic fraction of HUVEC possesses 3xe2x80x2-exo-nucleolytic activity which is responsible for degradation of natural and PS-oligomers. The enzyme is Rp-specific, i.e. it cleaves internucleotide phosphorothioate function of Rp- and not Sp-configuration at phosphorus atom.
Enzymatic hydrolysis of stereoregular oligodeoxyribonucleoside phosphorothioates (PS-oligos) synthesized via the oxathiaphospholane method has been used for assignment of their diastereomeric purity (Koziolkiewicz et al., Antisense Nucleic Acid Drug Dev., 1999, 9, 171-181). For this purpose, two well-known enzymes of established diastereoselectivity, nuclease P1 and snake venom phospho-diesterase (svPDE) have been used. However, because of some disadvantageous properties of svPDE, a search for other [Rp]-specific endonucleases was undertaken. Extracellular bacterial endonuclease isolated from Serratia marcescens accepts PS-oligos as substrates and hydrolyzes phosphorothioate bonds of the [Rp] configuration, whereas internucleotide [Sp]-phosphorothioates are resistant to its action. Cleavage experiments carried out with the use of unmodified and phosphorothioate oligonucleotides of different sequences demonstrate that the Serratia nuclease is more selective in recognition and hydrolysis of oligodeoxyribonucleotides than previously reported. The substrate specificity exhibited by the enzyme is influenced not only by the nucleotide sequence at the cleavage site but also by the length and base sequence of flanking sequences. The Serratia nuclease can be useful for analysis of diastereomeric purity of stereodefined phosphorothioate oligonucleotides, but because of its sequence preferences, the use of this enzyme in conjunction with svPDE is more reliable.
The first NMR solution structure of a DNA/RNA hybrid containing stereoregular Rp-phosphorothioate modifications of all DNA backbone linkages is presented.
The complex of the enzymically synthesized phosphorothioate DNA octamer (all-Rp)-d(GCGTCAGG) and its complementary RNA r(CCUGACGC) had an overall conformation within the A-form family (Bachelin et al., Nat. Struct. Biol., 1998, 5, 271-276). Most helical parameters and the sugar puckers of the DNA strand assume values intermediate between A- and B-form. The close structural similarity with the unmodified DNA/RNA hybrid of the same sequence may explain why both the natural and the sulfur-substituted complex can be recognized and digested by RNase H.
New monomers, 5xe2x80x2-O-DMT-deoxyribonucleoside 3xe2x80x2-O-(2-thio-xe2x80x9cspiroxe2x80x9d-4,4-penta-methylene-1,3,2-oxathiaphospholane)s, were prepared and used for the stereo-controlled synthesis of PS-Oligos via the oxathiaphospholane approach (Stec et al., J. Am. Chem. Soc., 1998, 120, 7156-7167). These monomers and their 2-oxo analogs were used for the synthesis of xe2x80x9cchimericxe2x80x9d constructs (PS/PO-Oligos) possessing phosphate and P-stereo-defined phosphorothioate inter-nucleotide linkages. The yield of a single coupling step is approximately 92-95%, and resulting oligomers are free of nucleobase- and sugar-phosphorothioate backbone modifications. Thermal dissociation studies showed that for hetero-duplexes formed by [Rp]-, [Sp]-, or [mix]-PS/PO-T10 with dA12, dA30, or poly(dA), for each template, the melting temperatures as well as free Gibbs"" energies of dissociation process, are virtually equivalent. Stereochemical evidence derived from crystallographic analysis of one of the oxathiaphospholane monomers strongly supports the participation of pentacoordinate intermediates in the mechanism of the oxathiaphospholane ring-opening condensation.
The DBU-assisted 1,3,2-oxathiaphospholane ring opening condensation of the separate diastereomers of 5xe2x80x2-O-DMT-2xe2x80x2-O-TBDMS-ribonucleoside-3xe2x80x2-O-(2-thiono-1,3,2-oxathiaphospholane)s with 2xe2x80x2-TBDMSi-protected ribonucleoside bound to the solid support via the 3xe2x80x2-oxygen occurs with 96-100% stereospecificity and gives, after deprotection, [Rp]- or [SP]-diribonucleoside 3xe2x80x2,5xe2x80x2-phosphorothioates I (B=adenine, cytosine, guanine, uracil) in 65-97% yield (Sierzcha-la et al., J. Org. Chem., 1996, 61, 6713-6716). Attempts to improve these yields by increasing either the coupling time or DBU concentration were unsuccessful. The absolute configuration at phosphorus of the dimers (I) was assigned by treatment with the stereospecific nucleases snake venom PDE or nuclease P1. Discrimination of [Rp]- vs [Sp]-diastereomers of the following dimer by nuclease P1 is much less profound than that observed for dideoxyribonucleoside 3xe2x80x2,5xe2x80x2-phosphorothioates. 
Diastereomerically pure 5xe2x80x2-O-DMT-nucleoside 3xe2x80x2-O-(2-thio-1,3,2-oxathiaphospholanes) (B=T, Adebz, Cytbz) were used for the synthesis of stereo-regular phosphorothioates (Stec et al., J. Am. Chem. Soc., 1995, 117, 12019-12029). The oxathiaphospholane ring-opening condensation requires the presence of strong organic base, preferably DBU. The yield of a single coupling step is ca. 95% and resulting S-Oligos are free of nucleobase- and sugar-phosphorothioate backbone modifications. The diastereomeric purity of products was estimated on the basis of diastereoselective degradation with Nuclease P1 and a mixture of snake venom phosphodiesterase and Serratia marcescens endonuclease. Thermal dissociation studies of hetero-duplexes phosphorothioates/DNA and phosphorothioates/RNA showed that their stability is stereochemical- and sequence-dependent. 
It has been previously reported that four membered cyclic sulfur compounds are kinetically and thermodynamically facile compounds to form (Eliel et al., J. Am. Chem. Soc., 1985, 107, 2946-2952). A combination of product and rate studies including Hammett LFER for k and ks for p-substituted 3-(arylthio)-3-methyl-1-Bu tosylates and the solvent and salt effects on product ratio indicate that anchimeric assistance in the solvolysis of branched 3-(alkylthio) and (3-arylthio)propyl tosylates is real and that a marked Thorpe-Ingold effect is evident. This observation led us to design compounds shown in FIGS. 2 to 7 as chiral auxillaries to synthesize chiral phosphorothioates. In a similar publication the neighboring group participation of oxygen in the solvolysis of acyclic-alkoxy substituted p-toluenesulfonates was illustrated (Eliel et al., J. Org. Chem, 1985, 50, 2707-2711). Methanolysis of PhCH2OCRR1CR2R3CHR4OTs (R=Me, R1-R4=H; R=R1=Me, R2-R4=H; R=R1=R4=Me, R2=R3=H; R=R1=R3=R4=H, R2=Me; R=R1=R4=H, R2=R3=Me; Ts=O2SC6H4Me-p) proceeds with partial rearrangement, implying neighboring-group participation, only when there are geminal Me groups in the 2xe2x88x92 or 3xe2x88x92-position (R2=R3=Me or R=R1=Me).
In a recent review article entitled xe2x80x9cNew gem- and vic-disubstituent effects on cyclizationsxe2x80x9d, (Jung, Michael E., Synlett, 1999, 843-846 a summary of several new gem-disubstituent effects on cyclizations are illustrated, e.g., the gem-dialkoxy, -dicarboalkoxy, and -dithioalkoxy effects, have been discovered. In addition they have also observed a new vicinal disubstituent effect. A novel ring size effect of ketals on radical cyclizations has been investigated. In a similar article by the same author it was disclosed that while reaction of the bromoalkene with a 5-membered ketal I (R=Br, n=1) with tributyltin hydride gave only the acyclic product I (R=H, n=1), reaction of the corresponding bromoalkene with a 6-membered ketal I (R=Br, N=2) gave good yields of the cyclobutane II, in a novel ketal ring size effect. Also the gem-dicarboalkoxy effect was operative in these systems, e.g., cyclization of the bromo alkene triester, (E)-MeO2CCH:CHCH2C(CO2Et)2CH2OC(:S)OPh, afforded reasonable yields of the cyclobutane III. 
In accordance with this theory, the structures 3, 8, 14, 18, 20, and 25 all have geminal disubstituents. Use of this concept to synthesize chiral phosphorothioates with the concurrent formation of 4-membered cyclic thio compounds is novel.
Oligonucleotides that have chiral Sp phosphorothioate internucleotide linkages at the 3xe2x80x2-terminus are disclosed in International Application WO 99/05160, published by the PCT Feb. 4, 1999.
The present invention provides nuclease resistant phosphorothioate oligonucleotides which are useful for therapeutics, diagnostics and as research reagents. In preferred embodiments, the invention provides oligomeric compounds comprising a plurality of covalently-bound nucleosides, which have the formula:
5xe2x80x2-T1xe2x80x94(Nuxe2x80x94Sp)nxe2x80x94(Nuxe2x80x94Lp)mxe2x80x94(NUxe2x80x94Sp)pxe2x80x94Nuxe2x80x94T2-3xe2x80x2
wherein:
T1 and T2 are each, independently, hydroxyl, a protected hydroxyl, a covalent attachment to a solid support, a nucleoside, an oligonucleoside, a nucleotide, an oligonucleotide, a conjugate group or a 5xe2x80x2 or 3xe2x80x2 substituent group;
each Sp is a chiral Sp phosphorothioate internucleoside linkage;
each Lp is, independently, a chiral Rp phosphorothioate internucleoside linkage, a racemic phosphorothioate internucleoside linkage or an internucleoside linkage other than a chiral phosphorothioate internucleoside linkage.;
each n and m is, independently, from 1 to 100;
each p is from 0 to 100; where the sum of n, m and p is from 3 to about 200;
each Nu independently, has the formula: 
xe2x80x83wherein:
Bx is a heterocyclic base moiety; and
R1 is H, hydroxyl, a protected hydroxyl, a 2xe2x80x2-substituent group or a protected 2xe2x80x2-substituent group.
In some preferred embodiments, each R1 is H or hydroxyl. In further preferred embodiments, R1 is C1-C10 O-alkyl or C1-C10 substituted O-alkyl, with 2xe2x80x2-O-methoxyethyl or 2xe2x80x2-O-methyl being moire preferred.
In some preferred embodiments, each Nu is, independently, adenosine, guanosine, uridine, 5-methyluridine, cytidine, 5-methylcytidine or thymine.
In some more preferred embodiments, p is 1 or 2. In further more preferred embodiments, n and p are each 1 and m is from 3 to about 20.
In some preferred embodiments, T1 and T2 are, independently, hydroxyl or a protected hydroxyl. In further preferred embodiments, each Lp is an Rp phosphorothioate internucleoside linkage. In still further preferred embodiments, at least one Lp is a racemic phosphorothioate internucleoside linkage. In still further preferred embodiments, at least one Lp is an internucleoside linkage other than a chiral phosphorothioate internucleoside linkage.
In some preferred embodiments, R1 is a 2xe2x80x2-substituent group or a protected 2xe2x80x2-substituent group other than H, hydroxyl or a protected hydroxyl.
The present invention also provides compounds having the formula: 
wherein:
Bx is a heterocyclic base moiety;
R4 is a hydroxyl protecting group;
R1 is H, hydroxyl, a protected hydroxyl, a 2xe2x80x2-substituent group or a protected 2xe2x80x2-substituent group; and
R2 is an Sp chiral auxiliary group.
In some preferred embodiments, the chiral auxiliary group has one of formulas I, II, III, IV, V or VI: 
In further preferred embodiments, Bx is adenosine, guanosine, uridine, 5-methyluridine, cytidine, 5-methylcytidine or thymine.
In further preferred embodiments, each R1 is H or hydroxyl. In still further preferred embodiments, R1 is C1-C10 O-alkyl or C1-C10 substituted O-alkyl, with 2xe2x80x2-O-methoxyethyl or 2xe2x80x2-O-methyl being more preferred.
In some preferred embodiments, at least one R1 is 2xe2x80x2-O-methoxyethyl or 2xe2x80x2-O-methyl. In further preferred embodiments, R1 is a 2xe2x80x2-substituent group or a protected 2xe2x80x2-substituent group other than H, hydroxyl or a protected hydroxyl.
The present invention also provides pharmaceutical compositions comprising one or more compounds of the invention, and an acceptable pharmaceutical carrier.
The present invention also provides methods for preparing an oligomeric compound of formula:
5xe2x80x2-T1xe2x80x94(Nuxe2x80x94Sp)nxe2x80x94(Nuxe2x80x94Lp)mxe2x80x94(NUxe2x80x94Sp)pxe2x80x94Nuxe2x80x94T2-3xe2x80x2
wherein:
each T1 and T2 is, independently, hydroxyl, a protected hydroxyl, a covalent attachment to a solid support, a nucleoside, an oligonucleoside, a nucleotide or an oligonucleotide, a conjugate group or a 5xe2x80x2 or 3xe2x80x2 substituent group;
each Sp is an Sp phosphorothioate internucleoside linkage;
each Lp is, independently, an Rp phosphorothioate internucleoside linkage, a racemic phosphorothioate internucleoside linkage or an internucleoside linkage other than a chiral phosphorothioate internucleoside linkage;
each n and m is, independently, from 1 to 100;
each p is from 0 to 100 where the sum of n, m and p is from 3 to about 200;
each Nu, independently, has the formula: 
xe2x80x83wherein:
Bx is a heterocyclic base moiety; and
R1 is H, hydroxyl, a protected hydroxyl, a 2xe2x80x2-substituent group or a protected 2xe2x80x2-substituent group;
comprising the steps of:
(a) providing a compound of formula: 
xe2x80x83wherein:
R4 is a labile hydroxyl protecting group;
R3 is a covalent attachment to a solid support;
(b) deblocking said labile hydroxyl protecting group to form a deblocked hydroxyl group;
(c) optionally treating said deblocked hydroxyl group with a further compound having the formula: 
xe2x80x83wherein:
R2 is an Sp chiral auxiliary group;
and a condensing reagent to form an extended compound;
(d) optionally repeating steps (b) and (c);
(e) treating said deblocked hydroxyl group with a compound having the formula: 
xe2x80x83wherein:
R5 is an Rp chiral auxiliary group or an activated phosphorus group;
and a condensing reagent to form a further extended compound;
(f) optionally repeating steps (e) and (f) to add further nucleosides;
(g) deblocking said labile hydroxyl protecting group to form a deblocked hydroxyl group;
(h) treating said deblocked hydroxyl group with a further compound having the formula: 
xe2x80x83wherein:
R2 is an Sp chiral auxiliary group;
and a condensing reagent to form a protected oligomeric compound; and
(i) optionally repeating steps (h) and (i) to add at additional nucleosides thereby forming a further protected oligomeric compound.
Preferably, the method further comprises the step of deblocking the product of step (i).
In some preferred embodiments of the methods of the invention, the Sp chiral auxiliary group has one of formulas I, II or III: 
or one of formulas IV, V or VI: 
More preferred embodiments of the methods of the invention, further comprise one or more capping steps, which include treatment with a capping agent. Preferably, such capping steps are performed after a coupling step, e.g., one or more of steps c, d, e, f, h, and/or i.
In some preferred embodiments, the methods of the invention further comprising one or more oxidation steps; said oxidation steps comprising treatment with an oxidizing agent. In some preferred embodiments, such oxidiation steps are performed after a coupling step, e.g., one or more of steps c, d, e, f, h, and/or i.
In some preferred embodiments of the methods of the invention, said labile hydroxyl protecting group is dimethoxytrityl, monomethoxy trityl, trityl or 9-phenyl-xanthene. In further preferred embodiments of the methods of the invention, said heterocyclic base moiety is a purine or a pyrimidine, which is preferably, independently, adenosine, guanosine, uridine, 5-methyluridine, cytidine, 5-methylcytidine or thymine.
In some preferred embodiments of the compounds and methods of the invention, the sum of n, m, and p is from 5 to about 50, with 8 to about 30 being more preferred, and with 10 to about 25 being even more preferred.
In further preferred embodiments of the methods of the invention, T1 and T2 are, independently hydroxyl or a protected hydroxyl.
In still further preferred embodiments of the methods of the invention, each Lp is a racemic phosphorothioate internucleoside linkage.
In still further preferred embodiments of the methods of the invention, at least one Lp is a racemic phosphorothioate internucleoside linkage.
In some more preferred embodiments, of the methods of the invention, n and p are each 1 and m is from 3 to about 20. In further more preferred embodiments n and p are each 2 and m is from 3 to about 20.
In further preferred embodiments, p is 0.
In some preferred embodiments, at least one R, is a 2xe2x80x2-substituent group or a protected 2xe2x80x2-substituent group other than H, hydroxyl or a protected hydroxyl.
In further preferred embodiments, the activated phosphorus group is a phosphoramidite, an H-phosphonate or a phosphate triester.
In still further preferred embodiments, the covalent attachment to a solid support is a sarcosinyl-succinonyl linker.
In further preferred embodiments, compounds are provided having the formula: 
wherein:
R62 is H or a hydroxyl protecting group;
R1 is H, hydroxyl, a protected hydroxyl, a 2xe2x80x2-substituent group or a protected 2xe2x80x2-substituent group;
B is a heterocyclic base moiety; and
R62 is a chiral auxiliary selected from formulas I-VI: 
Also provided in accordance with the invention are compounds having the formula: 
wherein:
q is 0 to about 50;
R62 is H or a hydroxyl protecting group;
R1 is H, hydroxyl, a protected hydroxyl, a 2xe2x80x2-substituent group or a protected 2xe2x80x2-substituent group;
R64 is H, a hydroxyl protecting group, or a linker to a solid support;
R63 is a radical selected from the group consisting of 
In some preferred embodiments, each R1 is H or hydroxyl. In further preferred embodiments, R1 is C1-C10 O-alkyl or C1-C10 substituted O-alkyl, with 2xe2x80x2-O-methoxyethyl or 2xe2x80x2-O-methyl being preferred.
In some preferred embodiments, B is independently, adenine, guanidine, uridine, 5-methyluridine, cytidine, 5-methylcytidine or thymine.
In further preferred embodiments, q is 5 to about 50, with 8 to about 30 being preferred, and 10 to about 25 being more preferred. In some particularly preferred embodiments, q is 0 or 1.
Also provided by the present invention are methods of modulating the production or activity of a protein in an organism, comprising contacting said organism with a compound of the invention, and methods of treating an organism having a disease characterized by the undesired production of a protein, comprising contacting said organism with a compound of the invention.
The present invention further provides methods of assaying a nucleic acid, comprising contacting a solution suspected to contain said nucleic acid with a compound of the invention.